CN112986335B - Process for fixing high molecular substance on chip - Google Patents
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- CN112986335B CN112986335B CN202110516707.8A CN202110516707A CN112986335B CN 112986335 B CN112986335 B CN 112986335B CN 202110516707 A CN202110516707 A CN 202110516707A CN 112986335 B CN112986335 B CN 112986335B
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Abstract
The invention relates to the field of biochips, and discloses a process for fixing high molecular substances on a chip, which comprises a chip activation step, a chip film forming step and a chip crosslinking step, wherein the chip comprises an electrode plate; in the chip activation step, air is used as a medium to carry out plasma cleaning on the chip to obtain an activated chip; in the chip film formation step, APTES was coated on the electrode sheet to obtain a film-formed chip. The invention can well fix the biological molecules on the chip, particularly well fix the biological molecules on the electrode plate of the chip, so that the chip can be used in a biological chip detection method based on electrical detection, and the development of the biological chip detection method based on the electrical detection is promoted. In addition, the finished chips produced in the same batch have stable quality and good repeatability.
Description
Technical Field
The invention relates to the field of biochips, in particular to a process for fixing a high molecular substance on a chip.
Background
The biochip integrates biochemical analysis process on the surface of the biochip according to the principle of specific interaction between biomolecules, thereby realizing high-flux rapid detection of DNA, polypeptide, protein and other biological components. The term "biochip" as used herein refers to a biomolecule array formed by immobilizing biomolecules (oligonucleotides, cDNAs, gDNAs, polypeptides, antibodies, antigens, etc.) on a solid material such as a silicon wafer, a glass plate, a plastic plate, a gel, or a nylon membrane by various methods. In the conventional biochip detection method, specific binding between a target biomolecule and a corresponding biomolecule coated on a biochip simply depends on diffusion motion and random brownian motion, and thus, the detection time is long. In order to shorten the time of biochip detection and improve the biochip detection efficiency, a biochip detection method based on electrical detection is proposed, wherein an electrode plate is arranged on a biochip, alternating current is provided for the electrode plate, a dielectrophoresis effect and an electrothermal effect are generated in a reaction unit, the combination of target biomolecules and corresponding biomolecules coated on the biochip is accelerated, the detection time is shortened, the detection efficiency is improved, and whether the target biomolecules are combined on the biochip is represented through the change of impedance values of the biochip.
Although the efficiency of the detection method of the electrical biochip is high, since the biochip uses the electrode sheet as a solid phase material, biomolecules cannot be well fixed on the electrode sheet, and thus how to well fix the biomolecules on the electrode sheet becomes a problem.
Disclosure of Invention
The invention aims to provide a process for fixing a high molecular substance on a chip so as to solve the problem of how to fix biomolecules on an electrode plate.
In order to achieve the purpose, the invention adopts the following technical scheme: a process for fixing high molecular substances on a chip comprises a chip activation step, a chip film forming step and a chip crosslinking step, wherein the chip comprises an electrode plate; in the chip activation step, air is used as a medium to carry out plasma cleaning on the chip to obtain an activated chip; in the chip film formation step, APTES was coated on the electrode sheet to obtain a film-formed chip.
The principle and the advantages of the scheme are as follows: in this scheme, since the chip includes the electrode sheet and is subsequently applied to the electricity-based biochip detection method, the biomolecules need to be immobilized on the electrode sheet. However, the solid phase material of the conventional biochip is generally poor or completely non-conductive substances such as silicon wafers, glass sheets, plastic sheets, gels, nylon films and the like, so that the common biomolecule fixing method is not suitable for electrode plates. Therefore, in the scheme, in the step of activating the chip, air is used as a medium to carry out plasma cleaning on the chip, so that hydroxyl groups, ketone groups, carboxyl groups, amino groups and other groups are modified on an electrode sheet of the chip, APTES (3-aminopropyltriethoxysilane) is combined, the APTES is subjected to hydroformylation modification after film formation on the chip and is combined with biomolecules, so that the biomolecules are fixed on the chip, the problem of how the biomolecules are fixed on the electrode sheet is solved, and the difference variation coefficient in a chip coating batch is controlled within the range of 11.2-13.0%, so that the difference of the biomolecular weights fixed on finished chips produced in the same batch is reduced, and the quality of the finished chips produced in the same batch is more stable.
Preferably, as an improvement, in the chip activation step, the vacuum degree of the plasma cleaning machine is 0.3-0.5mbar, the power is 50-200w, and the cleaning time is 5-15 min.
Has the advantages that: the vacuum degree and the power of the plasma cleaning machine are limited, the cleaning time is in the range, the chip can be better cleaned, corresponding groups are modified on the chip, and the next procedure is guaranteed to be carried out efficiently.
Preferably, as an improvement, in the step of forming the film on the chip, the activated chip is soaked into an ethanol solution of 1-10wt% of APTES, and the APTES is coated on the electrode sheet.
Has the advantages that: in the scheme, when the concentration of the APTES is 1-10wt%, the film forming effect of the chip is better, so that the quality of finished chips produced in the same batch is more stable.
Preferably, as an improvement, in the step of cross-linking the chip, the chip after film formation is heated and solidified, 1-10wt% glutaraldehyde pure water solution is dripped after cooling, the chip is rinsed with ultrapure water after being placed for 0.5-2.0h, and the chip after cross-linking is obtained by blowing dry with nitrogen.
Has the advantages that: and aldehyde group modification is carried out on the film-formed chip by utilizing a glutaraldehyde pure water solution so as to be capable of combining and crosslinking corresponding biomolecules in the following process.
Preferably, as an improvement, before the chip activation step and before the chip cross-linking step, the chip is subjected to microscopic examination: observing the surface of the chip by using a metallographic microscope, and taking the chip without broken strips, connecting strips and adhering impurities.
Has the advantages that: before the step of activating the chip and before the step of cross-linking the chip, the chip is subjected to microscopic examination, so that unqualified chips are removed, and useless work is avoided. Wherein, the basis for judging that no adhering impurities exist on the surface of the chip is as follows: the interdigital part of the electrode plate has no spot larger than 0.5 mu m, and if the spot, the dirt and the dust particles are judged to be unqualified.
Preferably, as an improvement, in the step of cross-linking the chip, the curing temperature is 50-100 ℃ and the curing time is 30-120 min.
Has the advantages that: when the curing temperature and the curing time in the chip cross-linking step are limited within the above ranges, the aldehyde modification of the APTES film on the subsequent chip is facilitated.
Preferably, as an improvement, in the step of cross-linking the chip, after the glutaraldehyde pure water solution is dropped, the chip is placed in a moisture-preserving box at 18-25 ℃, and the humidity of the moisture-preserving box is 40-60%.
Has the advantages that: the chip is put into a moisture preservation box with the humidity of 40-60% for aldehyde modification, which is more beneficial to aldehyde modification of the APTES film on the chip.
Preferably, as an improvement, the method further comprises a chip coating step, wherein in the chip coating step, a biomolecule solution is dripped on the crosslinked chip, and the chip is incubated at 18-25 ℃ for 2-24h to obtain the coated chip.
Has the advantages that: in the chip coating step, biomolecules are bound to the chip after the hydroformylation modification, thereby immobilizing the biomolecules on the chip.
Preferably, as an improvement, in the chip coating step, the solvent of the biomolecule solution is a boric acid buffer solution, and the preparation method of the boric acid buffer solution is as follows: adding 0.0125-0.05M sodium tetraborate solution into 0.05-0.2M boric acid solution until pH is 5-8.
Has the advantages that: in the step of coating the chip, the boric acid buffer solution is used as a solvent of a biomolecule solution, so that the corrosion of the electrode plate can be effectively delayed, and the effective storage period of the chip is prolonged.
Preferably, as an improvement, in the step of forming the film on the chip, the soaking time is 5-60min, and after soaking, the chip is washed by absolute ethyl alcohol and dried by nitrogen.
Has the advantages that: the time for soaking the chip in the APTES solution is limited, and the influence on the quality of the chip caused by too long soaking time is avoided. And meanwhile, after soaking, the chip is washed by absolute ethyl alcohol, so that the time for drying by nitrogen is shortened.
Detailed Description
The following is further detailed by way of specific embodiments:
example 1
A process for fixing high molecular substance on chip includes such steps as activating chip, filming, cross-linking and coating, and includes providing an electrode plate, reaction unit of Chinese patent CN104965081B with the name of "antibody-antigen detection method based on mobile device", including a reaction cavity with open top, detecting plate under said reaction cavity, and at least a pair of electrode plates on said detecting plate. In this embodiment, the structure of the detection plate with the electrode pads laid thereon can be referred to the inventor's prior published papers (Development of an AC electronics-based imaging system for on-site diagnostics of electrical diseases, Xiaozhu Liu, Sensors and Actuators a, 171 (2011) 406-413, fig. 3 (b)). Usually, the reaction chamber and the detection plate are made of silicon (si), and the electrode plate is made of metal (aluminum, gold or copper, in this embodiment, aluminum).
1) Step of pretreatment
Before the chip activation step, performing primary microscopic examination on the chip, observing the surface of the chip under a 10-time ocular by using a metallographic microscope, taking the chip without broken strips, continuous strips or adhering impurities, and rejecting unqualified chips. In the process, the basis for judging that no adhering impurities exist on the surface of the chip is as follows: the interdigital parts of the electrode plates (namely gaps among the electrode plates) have no spots, particles, dirt and dust particles larger than 0.5 mu m, and if the spots, the dirt and the dust particles are judged to be unqualified.
2) Chip activation step
And (2) using air as a cleaning medium, and performing plasma cleaning on the chip by using a plasma cleaning machine in the prior art, wherein the vacuum degree of the plasma cleaning machine is 0.5mbar (optional range of 0.3-0.5 mbar), the power is 50w (optional range of 50-200 w), the cleaning time is 10min (optional range of 5-15 min), and the activated chip is obtained. The purpose of the step is to carry out surface cleaning and modification on the surface of the chip, and oxygen in the air is used for generating groups such as-OH, -C = O, -COOH and the like on the surface through oxidation reaction; and using nitrogen in the air to generate-NH on the surface of the chip2A group.
3) Chip film formation step
Soaking the activated chip into an ethanol solution of 10wt% of APTES (the APTES is dissolved in absolute ethyl alcohol, the mass fraction of the APTES is 10%, and the APTES is 3-aminopropyltriethoxysilane), soaking for 30min (the optional range is 5-60 min) at normal temperature (18-25 ℃ in terms of normal temperature), washing each chip with absolute ethyl alcohol for 30s, and drying with nitrogen to obtain the film-formed chip.
And performing secondary microscopic examination on the film-formed chips, observing the surfaces of the chips by using a metallographic microscope under a 10-time eyepiece, photographing and recording the surface condition of each chip, taking chips without broken strips or adhesive impurities, and rejecting unqualified chips. In the process, the basis for judging that no adhering impurities exist on the surface of the chip is the same as the first microscopic examination.
4) Chip cross-linking step
Heating and curing the film-formed chip at 63 deg.C (optional range of 50-100 deg.C) for 60min (optional range of 30-120 min). After natural cooling, 10 mu L of 2.5wt% glutaraldehyde solution (prepared by pure water) is dripped into the reaction cavity of the chip, the glutaraldehyde solution covers the electrode plate, and then the chip is placed in a moisture preservation box for 1h (with the optional range of 0.5-2.0 h) at 22 ℃ (with the optional range of 18-25 ℃), and the humidity of the moisture preservation box is 40% (with the optional range of 40-60%). Then, the chip is washed by ultrapure water for 10s, and dried by nitrogen to obtain the crosslinked chip.
5) Chip coating step
And (3) dropwise adding a biomolecule solution onto the cross-linked chip, and incubating at 22 ℃ (optional range of 18-25 ℃) for 20h (optional range of 2-24 h) to obtain the coated chip. In this example, 10 μ L of commercial brucella omp antigen (BBS) was added dropwise to the crosslinked chip, wherein the antigen was dispersed and dissolved using a Borate Buffer (BBS) as a solvent, and the preparation method of the borate buffer was: adding 0.0125-0.05M sodium tetraborate solution into 0.05-0.2M boric acid solution until pH is 5-8. In this example, the preparation method of the borate buffer (i.e., 100mM BBS) was as follows: to a 0.1M boric acid solution was added a 0.025 sodium tetraborate solution to a pH of 7.4. In this example, different antigens can be selected according to the condition of the biomolecule to be detected.
In the chip coating step, after the chip is incubated for 5min, an impedance instrument is used for carrying out impedance detection on the chip, specifically, a group of antibodies is used for being connected to a terminal of an electrode plate to carry out impedance frequency scanning measurement and analysis, the scanning frequency range is 1MHz to 100Hz, the excitation voltage is 5mV (the selectable range is 1mV-100 mV), the number of sampling points is 201, the measurement time is 3s, and impedance frequency scanning data of each chip are stored (namely impedance scanning before coating, response parameters of the electrode plate under different scanning frequencies, such as impedance, phase, resistance components, capacitance components, inductance components and the like, are obtained, and a curve of the parameters changing along with the frequency before coating is obtained by drawing).
After the impedance scanning before coating is finished, the chip is put in a humidity box and incubated for 20h (optional range of 2-24 h) at 22 ℃ (optional range of 18-25 ℃) to obtain the coated chip. After coating, taking out the chip from the moisture preservation box, then detecting the impedance of the coated chip by using an impedance meter, similarly, scanning frequency ranges from 1MHz to 100Hz, excitation voltage is 5mV (the optional range is 1mV-100 mV), the number of sampling points is 201, measuring time is 3s, and storing impedance frequency scanning data of each chip (called as post-coating impedance scanning, obtaining response parameters of the electrode slice under different scanning frequencies, such as impedance, phase, resistance component, capacitance component, inductance component and the like, and drawing to obtain a curve of the parameters changing along with the frequency before coating).
Comparing the impedance scanning results before coating and after coating to judge whether the chip is qualified, wherein the judging method comprises the following steps: under the impedance scanning frequency, calculating the change rate of the capacitance value obtained by scanning after coating and the capacitance value obtained by scanning before coating, wherein the specific calculation method comprises the following steps: capacitance change rate = (capacitance value of scan after coating-capacitance value of scan before coating)/capacitance value of scan before coating × 100%. The capacitance change rate needs to be controlled within a range of-50.0-150.0%, and chips which are not within the range are unqualified chips and need to be discarded. The specific impedance scanning frequency is confirmed through a curve of capacitance changing along with frequency before coating and a curve of capacitance changing along with frequency after coating, and the specific confirmation method comprises the following steps: and calculating to obtain the capacitance change rate under the same frequency of pre-coating scanning and post-coating scanning, wherein the scanning frequency value corresponding to the maximum value (if the value is a negative number, the absolute value is taken) of the capacitance change rate is the specific impedance scanning frequency. In the embodiment, the capacitance change rate is specifically calculated at a frequency of 50KHz (i.e., a specific impedance scanning frequency), and the capacitance change rate at the specific impedance scanning frequency is controlled within a range of 60.0-100.0%, wherein the capacitance change rate at the specific impedance scanning frequency is also referred to as a maximum capacitance change rate before and after scanning. Since different chip batches and coated biomolecules can cause different specific impedance scanning frequencies, impedance scanning needs to be performed before and after coating to obtain the specific impedance scanning frequency, and the yield of the finished chip can be ensured only if the capacitance change rate under the specific impedance scanning frequency needs to be maintained within a certain range.
And then observing the surface of the chip by using a metallographic microscope under a 10-fold ocular lens, photographing and recording the surface condition of each core, and abandoning the chip (called as third microscopic examination) if the surface is damaged or polluted seriously, wherein the judgment method is the same as the first microscopic examination.
6) Sealing and drying
Each coated chip was added 20. mu.L of 100mM BBS using a 200. mu.L pipette and then blown dry with nitrogen and repeated 1 time. Observing the surface of each chip by using a metallographic microscope under a 10-fold eyepiece, photographing and recording the surface condition of each chip, abandoning the chip (called fourth microscopic examination) if the surface is damaged or polluted seriously, and judging by the same method as the first microscopic examination. mu.L of 10% bovine serum albumin blocking solution (solvent 100mM BBS) was added dropwise using a 10. mu.L pipette and blocked at room temperature for 0.5 h. And adding 20 mu L of 100nM BBS into each chip by using a 200 mu L pipette, then blowing and drying by using ammonia gas, and repeating for 1 time to obtain the finished chip.
And (3) observing the surface of each finished chip by using a metallographic microscope under a 10-fold eyepiece, photographing and recording the surface condition of each chip, and if the surface is damaged or polluted seriously, abandoning the chip (called as fifth microscopic examination) and judging by the same method as the first microscopic examination.
Examples 2 to 4 and comparative examples 1 to 4 are basically the same as example 1 except for the points shown in table 1. In Table 1, "N/A" in comparative example 3 indicates that no chip activation step was performed, and "N/A" in comparative example 4 indicates that no chip film formation step was performed.
TABLE 1 setup of Process Steps and parameters in examples and comparative examples
| Medium selection in chip activation step | APTES concentration (%) | |
| Example 1 | Compressed air | 10 |
| Example 2 | Compressed air | 5 |
| Example 3 | Compressed air | 1 |
| Example 4 | Compressed air | 15 |
| Comparative example 1 | Nitrogen gas | 10 |
| Comparative example 2 | Oxygen gas | 10 |
| Comparative example 3 | N/A | 10 |
| Comparative example 4 | Compressed air | N/A |
Experiment one
For examples 1 to 4 and comparative examples 1 to 4, in each example and each comparative example, 20 finished chips are selected, under the specific impedance scanning frequency of each chip, the change rate of the capacitance value obtained by scanning after coating and the capacitance value obtained by scanning before coating of each chip is calculated, then the average value and the standard deviation of the capacitance change rate of the chip in each example and comparative example are calculated, the intra-coating-lot difference coefficient CV of each example and comparative example is calculated, and the calculation formula of the intra-coating-lot difference coefficient CV of the chip is as follows: coefficient of Variation (CV) — (standard deviation SD/average X) 100%. The variation coefficient of the difference between the chip coating batches in the examples and comparative examples is shown in Table 2.
TABLE 2 DIFFERENTIAL VARIATION COEFFICIENCY IN COATING BATTERIES FOR THE DIE-COATING OF THE DIE OF THE EXAMPLES AND THE COMPARATIVE RATIONS
| Example 1 | Example 2 | Example 3 | Example 4 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | |
| Coefficient of variation of inner difference CV (%) > of chip coating batch | 11.2 | 12.4 | 13.0 | 17.5 | 27.4 | 22.3 | 32.8 | 58.4% |
As can be seen from table 2, the coefficient of variation CV in the chip coating lot of example 1 is only 11.2%, and compared with the coefficient of variation in the chip coating lot of comparative example 4, it is clear that the formation of the APTES film on the chip significantly contributes to the stable immobilization of the biomolecules on the chip, and the biomolecules are not easily dropped off, so that the immobilized biomolecules on the finished chips produced in the same lot tend to be more uniform, and the stability of the quality of the finished chips produced in the same lot is ensured.
Comparing example 1 with the intra-chip coating lot difference coefficient of variation of comparative example 3, it is understood that the difference in the amount of biomolecules immobilized thereon is large in the finished chips produced without the chip activation step even when the finished chips are produced in the same lot, resulting in unstable quality of the finished chips produced in the same lot.
Comparing examples 1 to 4 with comparative examples 1 and 2, the difference coefficient of variation in the chip coating lot of examples 1 to 4 is smaller than that of comparative examples 1 and 2, which shows that the selection of cleaning medium in the chip activation step will affect the subsequent chip processing step. In the invention, when air is selected as a cleaning medium, the internal difference of the chip coating batch can be effectively reduced, and the quality stability of the finished chip is improved.
Comparing examples 1 to 4 internally, it is not easy to find that the concentration of APTES also affects the differential coefficient of variation within the chip coating lot in the step of forming the film on the chip, and experiments show that the differential coefficient of variation within the chip coating lot is 11.2 to 13.0% when the concentration of APTES is in the range of 1 to 10wt%, the differential coefficient of variation within the chip coating lot is small, and the stability of the quality of the finished chip is good.
In summary, in the present invention, air is used as a cleaning medium for plasma cleaning, an APTES film is formed on a chip, and the concentration of APTES in the step of forming a film on the chip is limited to 1-10wt%, so as to reduce the difference coefficient of variation within a chip coating lot, so that the biomolecular weights fixed on finished chips produced in the same lot tend to be the same, and the stability of the quality of the finished chips in the same lot is improved, thereby the repeatability of the finished chips in the same lot is good.
Experiment two
Examples 5 to 10 and comparative examples 5 to 8 are basically the same as example 1 except that the buffers selected in "5) the chip coating step" and "6) the blocking and drying steps" are different, and the specific differences are shown in Table 3. And, the finished chips prepared in examples 5 to 10 and comparative examples 5 to 8 were placed in a dry sealed bag (10 finished chips were used in each example and comparative example, and were individually packaged in the dry sealed bag), the surface of the chip (mainly, electrode tab) was observed daily under a 10-fold eyepiece using a metallographic microscope, whether or not the corrosion phenomenon occurred in the electrode tab was judged, if so, the date of the occurrence of the phenomenon was recorded, and the duration of corrosion resistance was counted, and the results are shown in table 3.
TABLE 3 Corrosion duration test results (mean + -SD, N = 10)
| Test buffer type | Test buffer solution preparation method | Duration of tarnish resistance (Tian) | |
| Example 1 | BBS | See example 1 | 67.20±3.91 |
| Example 5 | BBS | Adding 0.0125M sodium tetraborate solution into 0.05M boric acid solution until pH is 5.0 | 63.10±3.60 |
| Example 6 | BBS | Adding 0.05M sodium tetraborate solution into 0.2M boric acid solution until pH is 8.0 | 62.90±3.70 |
| Example 7 | BBS | Adding 0.025M sodium tetraborate solution into 0.1M boric acid solution until pH is 9 | N/A |
| Example 8 | BBS | Adding 0.025M sodium tetraborate solution into 0.1M boric acid solution until pH is 4 | N/A |
| Example 9 | BBS | Adding 0.1M sodium tetraborate solution into 0.3M boric acid solution until pH is 7.4 | 55.20±2.70* |
| Example 10 | BBS | Adding 0.005M sodium tetraborate solution into 0.02M boric acid solution until pH is 7.4 | 52.70±3.13* |
| Comparative example 5 | PBS | See PBS formulation (pH7.4) | N/A |
| Comparative example 6 | Carbonic acid buffer | A0.1M sodium carbonate solution was added dropwise to a 0.1M sodium bicarbonate solution to a pH of 9.0. | N/A |
| Comparative example 7 | Boric acid solution | 0.1M boric acid solution | N/A |
| Comparative example 8 | Sodium tetraborate solution | 0.025M sodium tetraborate solution | N/A |
1L preparation of PBS (pH7.4) in Table 3: potassium dihydrogen phosphate 0.24 g; 1.44g of disodium hydrogen phosphate; 8g of sodium chloride; 0.2g of potassium chloride; adding deionized water about 800mL, stirring thoroughly to dissolve, adding concentrated hydrochloric acid to adjust pH to 7.4, and adding volume to 1L.
In table 3, it is shown that the experimental group has significant differences compared to the data of example 1 (T-test, p < 0.05). N/A indicates that the electrode pad had been largely corroded during chip processing (as found by microscopic examination), and belongs to a defective chip, and a satisfactory finished chip could not be efficiently obtained using the buffers (solutions) of example 7, example 8, and comparative examples 5 to 8.
As shown in Table 3, the finished chip prepared by the boric acid buffer solution of the present invention can have a longer shelf life, but if the concentration of boric acid or sodium tetraborate in the boric acid buffer solution is too high or too low, the pH value of the buffer solution is too high or too low, which is not favorable for forming an oxidation resistant film, and the obtained finished chip has a poor corrosion resistance effect. If other buffer solution is used, or the boric acid solution or the sodium tetraborate solution is used, the ideal anti-corrosion effect cannot be obtained.
The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.
Claims (5)
1. A process for fixing a high molecular substance on a chip is characterized in that: the method comprises a chip activation step, a chip film forming step, a chip crosslinking step and a chip coating step, wherein the chip comprises an electrode plate; in the chip activation step, air is used as a medium to carry out plasma cleaning on the chip to obtain an activated chip; in the step of chip film formation, soaking the activated chip into an ethanol solution of 1-10wt% of APTES, and covering the APTES on the electrode sheet to obtain a film-formed chip; the electrode plate is made of aluminum;
in the step of activating the chip, the vacuum degree of a plasma cleaning machine is 0.3-0.5mbar, the power is 50-200w, and the cleaning time is 5-15 min; in the step of chip cross-linking, the chip after film forming is heated and solidified, 1-10wt% glutaraldehyde pure water solution is dripped after cooling, ultra-pure water is washed after the chip is placed for 0.5-2.0h, and the chip after cross-linking is obtained by blowing dry with nitrogen; in the step of coating the chip, dripping a biomolecule solution on the crosslinked chip, and incubating for 2-24h at 18-25 ℃ to obtain the coated chip; the solvent of the biomolecule solution is boric acid buffer solution, and the preparation method of the boric acid buffer solution comprises the following steps: to a 0.1M boric acid solution was added a 0.025M sodium tetraborate solution to a pH of 7.4.
2. The process of claim 1, wherein the step of immobilizing the polymeric substance on the chip comprises: before the chip activation step and before the chip cross-linking step, performing microscopic examination on the chip: observing the surface of the chip by using a metallographic microscope, and taking the chip without broken strips, connecting strips and adhering impurities.
3. The process of claim 2, wherein the step of immobilizing the polymeric substance on the chip comprises: in the step of chip cross-linking, the curing temperature is 50-100 ℃, and the curing time is 30-120 min.
4. The process of claim 3, wherein the step of immobilizing the polymeric substance on the chip comprises: in the step of chip crosslinking, after the glutaraldehyde pure water solution is dripped, the chip is placed in a moisture preservation box at the temperature of 18-25 ℃, and the humidity of the moisture preservation box is 40-60%.
5. The process of claim 4, wherein the step of immobilizing the polymeric substance on the chip comprises: in the step of forming the film on the chip, the soaking time is 5-60min, and after soaking, the chip is washed by absolute ethyl alcohol and dried by nitrogen.
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| US7510942B2 (en) * | 2003-11-25 | 2009-03-31 | Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University | Molecular modifications of metal/dielectric interfaces |
| CN104730136A (en) * | 2015-03-26 | 2015-06-24 | 中国科学院上海微系统与信息技术研究所 | Biosensor based on germanium nanowire field effect transistor, method and application |
| CN105161437B (en) * | 2015-09-18 | 2017-12-08 | 北京工业大学 | The glass or the micro-structural alignment of quartz chip and pre- bonding method of plasmaassisted |
| KR20170052773A (en) * | 2015-11-04 | 2017-05-15 | 연세대학교 산학협력단 | Triboelectric generator and method for manufacturing the same |
| CN105789453B (en) * | 2016-03-28 | 2018-08-17 | 南昌大学 | A method of the small molecule of self assembly containing chlorine substituent and for improving electrode work content |
| CN108359577B (en) * | 2018-01-29 | 2022-03-04 | 燕山大学 | Zebra fish egg rotation micro-operation system based on micro-fluid drive and control method |
| CN108957007B (en) * | 2018-07-24 | 2021-08-17 | 无锡市人民医院 | Biosensor combining dialysis device and silicon nanowire field effect transistor |
| CN111122679B (en) * | 2019-12-25 | 2022-12-02 | 杭州电子科技大学 | DNA biosensor and preparation method and application thereof |
| CN111443072B (en) * | 2020-04-30 | 2023-12-22 | 苏州翊清环境科技有限公司 | Raman chip for virus detection, preparation method and virus rapid detection method |
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